Iron-Quercetin Complex Preconditioning Human Peripheral Blood Mononuclear Cells Accelerates Angiogenic and Wound Healing Ecacy

Background: Cells-based therapy is a highly promising treatment paradigm in ischemic disease due to its ability to repair tissues when implanted into a damaged site. These therapeutic effects have been involving a strong paracrine component resulting from the high levels of bioactive molecules they secrete in response to the local microenvironment. Therefore, the secreted therapeutic can be modulated by preconditioning the cells during in vitro culture. Herein, we investigated the potential use of magnetic resonance imaging (MRI) probes “Iron-Quercetin complex” or IronQ for preconditioning peripheral blood mononuclear cells (PBMCs) to expand proangiogenic cells and enhance their secreted therapeutic factors. Methods: PBMCs obtained from healthy donor blood were cultured in the presence of the Iron-Quercetin complex. Preconditioning-PBMCs differentiated cells were characterized by immunostaining. An enzyme-linked immunosorbent assay to describe the secreted cytokines. In vitro migration and tubular formation using human umbilical vein endothelial cells (HUVECs) were completed to investigate the proangiogenic ecacy. Results: IronQ signicantly increased mononuclear progenitor cells' proliferation and differentiation into the spindle-shape-like cells, expressing both hematopoietic and stromal cell markers. The expansion increased the number of colony-forming units (CFU-Hill). The conditioned medium obtained from IronQ-treated PBMCs contained a high level of Interleukin (IL)-8, IL-10, urokinase-type-plasminogen-activator (uPA), matrix metalloproteinases-9 (MMP-9), and tumor necrosis factor-alpha (TNF-α), and augmented migration and capillary network formation of HUVEC and broblast cells in vitro. Conclusions: Our study demonstrated that the IronQ-precondition PBMCs protocol could enhance the angiogenic and reparative potential of non-mobilized PBMCs. This protocol can be used as an adjunctive strategy to improve cell therapy's ecacy of PBMCs for ischemic diseases and chronic wound.


Introduction
Cell-based therapy has currently become a focus of regenerative treatment for ischemic diseases and chronic wounds. Clinical improvements have been observed using autologous total mononuclear cells (MNCs) freshly isolated from bone marrow or peripheral blood. These clinical experiences proved that cell-based therapy for vascular regenerations is safe, feasible, and effective [1]. However, the low e cacy of functional cells due to the lower number and dysfunction found in patients has limited its potential as a therapeutic tool [2]. In this study, we consider peripheral blood as a renewable cell source that can be retrieved from a readily accessible body compartment by a low-invasive procedure. However, the peripheral blood mononuclear cells (PBMCs) contain a rarity percentage (<0.01%) of stem/progenitor cells [3,4]. Moreover, aging or disease also attenuates the numerically and functionally of these functional cells [5]. Therefore, the biggest challenge lies in the enhanced cell expansion and cell therapeutic potential toward successful and wider clinical application using autologous cells while controlling costs.
Adult peripheral blood-derived stem/progenitor cells are of interest as a potential source of stem cells because they are known to transdifferentiate [6,7] and comprise the enriched fraction of endothelial progenitor cells (EPCs), which possess superior regenerative e cacy [8]. Moreover, peripheral bloodderived stem/progenitor cells can be obtained from autologous sources without the need for painful bone marrow aspiration. Although some clinical trials have shown potential bene ts of peripheral bloodderived stem cells in a patient with ischemic arterial disease [9][10][11][12], some controversial issues of the clinical bene ts of blood-derived stem cells have emerged. These might be due to the variety of cell isolation protocols and the type of cells administered to patients, such as either enriched progenitor fractions or whole mononuclear cell fractions.
According to the heterogeneity in the peripheral blood mononuclear cells population, our focus is on the critical functions of monocytes/macrophages. Monocytes are precursors of macrophages; monocytesderived progenitor cells play a crucial role in angiogenesis and regenerative processes. Monocyte-derived cells with spindle shape characteristics named brocytes, and endothelial progenitor cells, are proangiogenic potential in vitro and in vivo. Fibrocytes contribute to tissue repair and angiogenesis [13]. They are characterized by the expression of both hematopoietic and stromal cell markers (collagen I, collagen III, CD34, and CD45). Peripheral blood monocyte serves as an enriched source of endothelial progenitor cells. The involvement of EPCs in angiogenesis is well known. EPCs secreted angiogenic factors and differentiation into the endothelial cell [14]. These cells have been shown to affect vascular repair in ischemic hind limb models of vascular injury [15,16]. Moreover, monocyte cells can also transdifferentiate into endothelial [17], neuronal [18], and mature myeloid cells [19]. Many antigenic markers including, cluster of differentiation 34 (CD34), CD133, CD45, CD31, CD133, CD14, C-X-C Motif Chemokine Receptor 4 (CXCR4), and Vascular endothelial growth factor receptor-2 (VEGF-R2), have been utilized to identify EPC populations [20]. Depend on microenvironment stimulating, macrophages can be polarized into diverse subtypes that play different roles (i.e., the pro-in ammatory M1 phenotype and antiin ammatory M2 phenotype). Especially, the M2-subtype is playing a central stimulatory role within angiogenesis and tissue repair. These M2-like macrophages secrete an angiogenic factor to regulate vessel organization and vascular surveillance, as evidence by the irregular vessels in the absence of these macrophages [21,22].
Based on the rarity number of the therapeutic cells, to achieve clinically meaningful cell numbers and the superior quality of cultured cells. A practical approach based on peripheral blood progenitor cells mandates their ex vivo expansion is need. Interestingly, dietary compounds, which could promote the angiogenic potential, have been increasingly applied. Quercetin has been extensively studied due to its potential pharmacological properties and bene cial health effects [23]. In particular, quercetin has been reported a bene cial impact on cardioprotective [24]. It also was used as Chinese medicine for the treatment of heart disease [25]. Recent studies have reported that quercetin could enhance the cell proliferation, osteogenic differentiation, and angiogenic factor secretion of healthy rat bone marrow mesenchymal stem cells (BMSCs) [25]. Moreover, quercetin was also shown to modulate in ammation in humans through mechanisms involving macrophages by enhancing the anti-in ammatory properties of M2 macrophages [26]. However, poor water solubility, chemical instability, and low bioavailability of quercetin can signi cantly limit its clinical applications [27]. The complexation of quercetin and a large number of metal ions have been reported. It indicates that the biological activities can be improved and increased compared with free quercetin [28].
Recently, we have introduced a novel bi-functional magnetic resonance imaging (MRI) probe, the socalled Iron-Quercetin complex (IronQ) as a new MRI contrast agent for tracking labeled cells. IronQ modulated the cellular characteristic of PBMCs to enrich contained blood-derived spindle-shape-like cells (EPCs) during in vitro expansion. Simultaneously, there is the potential to visualize IronQ labeled cells transplant with MR imaging via the paramagnetic properties of IronQ [29]. The Iron-Quercetin complex has some practical advantages over other types of MRI contrast agents when used for clinical purposes.
(1) They can be ease prepared without the use of any toxic agents or expensive equipment. (2) They have non-toxicity to cells and can be enriched the circulating progenitor cells in expanded culture. (3)they have the potential to monitor by clinical magnetic resonance imaging. With the use of IronQ with conventional culture technique, blood-derived pro-angiogenic cells can be expanded, increasing their angiogenic potential while tracking IronQ labeled cells via MRI. To minimize the cost of the cell expansion method and the safety pro le of the cell tracking procedure, this IronQ was applied to PBMCs. The objective of this study was to examine the effect of the Iron-Quercetin complex on PBMC transforming on the change of surface markers and cytokine and growth factors released. Furthermore, we evaluated the therapeutic potential of expanded cells including, angiogenic and wound healing potentials. The IronQpreconditioning technique may enhance the isolated unstimulated peripheral blood progenitor cells' therapeutic potential for therapeutic vasculogenesis and tissue regeneration.

Ethics statement
Peripheral blood mononuclear cells (PBMCs) used in this study were obtained from healthy human peripheral blood (age 20 -40 years, n = 8), all donors provided informed consent. The study was approved by the Human Research Ethics Committee of the Faculty of Medicine, Chiang Mai University (ref. no. NONE-2560-05052).

Cell isolation and culture
PBMCs were obtained from a buffy coat bag from healthy donor volunteers at the Blood Bank Unit (Maharaj Nakorn Chiang Mai Hospital, Faculty of Medicine, Chiang Mai University). PBMCs were isolated from 100 mL of healthy blood donor buffy coat, both male and female. PBMCs were isolated over a Ficoll gradient (Lymphoprep™, Stemcell Technologies, Canada) using the manufacture's protocol. Mononucleated cell fractions were collected and cultured in RPMI 1640 medium with L-glutamine supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Human umbilical vein endothelial cells (HUVECs) were purchased from American Type Culture Collection (Manassas, VA, USA). HUVEC were cultured in an endothelial growth medium contained media DMEM/F-12 (10% FBS + 1% penicillin/streptomycin) supplement mix containing epidermal growth factor 5 ng/mL, basic broblast growth factor 10 ng/mL, insulin-like growth factor 20 ng/mL, vascular endothelial growth factor 0.5 ng/mL, heparin 22.5 µg/mL, hydrocortisone 0.2 µg/mL, and we de ned as EGM medium. L929 Mouse broblasts were purchased from American Type Culture Collection (Manassas, VA, USA). L929 were cultured in RPMI 1640 medium with L-glutamine supplemented with 10% FBS and 1% penicillin/streptomycin. Cultured all cells in an incubator at 37 ℃ humidi ed atmospheres with 5% CO 2 .

Iron-Quercetin complex (IronQ) treatment
Fresh PBMCs were seeded at a density of 1 × 10 6 cells/mL in RPMI 1640 (10% FBS and 1% penicillin/streptomycin) with or without 125 ug/mL Iron-Quercetin complex and cultured at 37 ℃ humidi ed atmospheres with 5% CO 2 . Experiments were performed after 7, 10, 14 days of culture without subculture or re-feeding. The changed in cell morphology was observed using an inverted microscope (Nikon, ECLIPSE Ts2, Tokyo, Japan).

Cell proliferation assay
The effect of IronQ on cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cells (1×10 6 cells/mL) were seed on 24-well plates in the presence of 125 µg/mL IronQ for 1, 3, 5, 7, and 10 days. At the end time point, 200 µL MTT (5 mg/mL) was added to each well and cells were further incubated for four h. Then removed the cultured supernatant and 500 µL of dimethyl sulfoxide (DMSO) was added. The intensity of the formazan solution was determined by measurement of the absorbance at 560 nm using a reader (BioTek TM Eon TM microplate reader, USA).
Preparation of PBMC conditioned medium PBMC cells were resuspended in RPMI 1640 medium at a density of 2×10 6 cells/mL and treated with 125 µg/mL IronQ for 10 Days (post-IronQ PBMC-CM). PMBC cells cultured in RPMI 1640 medium without IronQ used as control conditioned medium (Control PBMC-CM). After ten days of incubation, cells were further incubated with serum-free RPMI 1640 medium for 24 h. The cell suspension and conditioned medium were collected and centrifuged to eliminate cells and cellular debris and ltering by passing through a lter 0.22 µm pore size (Millex-Millipore™, Millipore Corporation, MA, USA).

Life cell labeling
The adherent post-IronQ treated PBMC cells were digested with 0.25% trypsin-EDTA and collected in phosphate buffer saline. According to the manufacturer's instructions, cells were labeled with green uorescence (PKH67 Fluorescent cell linker kits; Sigma-Aldrich, USA).

Tube formation assay on Matrigel
To investigate the angiogenic potential of post-IronQ PBMC cells. Matrigel (Geltrex TM , Life Technologies, USA) was used for in vitro tube formation assay. Brie y, Matrigel solution was added in 96-well plates at 37 °C for 1 h to allow the matrix solution to solidify. 1×10 4 cells HUVECs were seeded and cultured in 250 µL EGM medium or supernatant culture medium from pre-and-post-IronQ PBMCs. Where indicated, the post-IronQ PBMC cells were harvested and labeled with PKH67 uorescent cell linker and re-plated (5×10 3 cells/well), and co-cultured with HUVECs on the solidi ed matrix solution. Cells seeded on Matrigel were incubated at 37 °C. The tubule-like formation was observed under an inverted light microscope. To evaluate the angiogenic capacity, the total length of the tubes formed during the assay was analyzed by Wimasis Image Analysis (Cordoba, Spain). Post-IronQ treated PBMC-labeled cells incorporated into the tube formation were observed and captured using uorescence microscopy (Nikon, ECLIPSE Ts2, Tokyo, Japan).

HUVEC Migration with condition medium from post-IronQ treated PBMCs
HUVECs were serum-starved in EGM medium containing 2% FBS for 12 h. Then cells (1x10 5 cells) were seeded at the upper chamber of Transwell inserts of 24-well plates with an 8-mm membrane pore size (Corning, Life Sciences, USA.). The different conditioned medium was added as a chemoattractant in the lower chamber. After 12 h, the medium was aspirated, and the non-migrated cells in the upper surface of each membrane were removed by gently swabbed. Membranes were xed with methanol and stained with 0.5% crystal violet solution (Sigma-Aldrich, MO, USA). The stained cells were photographs from four random elds of view for each membrane under an inverted microscope (Nikon, ECLIPSE Ts2, Tokyo, Japan).
Scratch wound-induced broblast migration assay L929 broblasts cells were grown till they reached full con uence in 6-well plates and incubated overnight in a starvation medium. Cell monolayers were wounded with a sterile 200-µL pipette tip and washed with phosphate-buffered saline (PBS) to remove the detached cells from the plates. Cells were left untreated or treated with a conditioned medium and kept at 37 °C in a CO 2 incubator for 24 h. The wound gap was observed and photographed using phase-contrast microscopy (Nikon, ECLIPSE Ts2, Tokyo, Japan). The images were then analyzed using the Image J software 1.52v version (National Institutes of Health, USA) to measure the width of the scratch.

Statistical analysis
Data are expressed as the mean ± standard deviation (SD). The statistical analysis of mean comparison was performed by Student's t-test using OriginPro version 2018 program. Values of p ≤ 0.05 were considered statistically signi cant at the 5% signi cance level.

Results
PBMCs cultured under IronQ complex results in accelerating proliferative of adherent spindle-shaped cells and number of early outgrowth colonies (CFU-Hill) Firstly, the appropriate concentration of the Iron-Quercetin complex (IronQ) was determined by cytotoxicity assay in a previous study [29]. We found that 125 µg/mL of IronQ complex is not toxic to peripheral blood mononuclear cells (PBMCs) when cultured for a long time (i.e., for one month). These concentrations were also available for cell tracking by MRI. Then, PBMCs were cultured in RPMI 1640 medium with 10% FBS and 1% penicillin/streptomycin without adding any speci c growth factors, either in the absence (control) or in the presence of 125 µg/mL IronQ. Spindle shape cells appeared under both conditions with different characteristics. Under the IronQ condition, cells appear as long spindle cells considerable length of ~100 µm. Inversely, the majority population of attaching cells in the untreated control group appeared a shorter spindle and larger (Fig.1a). The total cell expansion of PBMCs cultured under the IronQ condition compare with the untreated control group was measured. The cell number in culture did not increase in the rst 3 days of culture but slightly increased at day 5. The number of cells gradually increased, from 1.3-fold on day 5 and up to 3-fold on day 14 after cells were treated with IronQ while the untreated control group number of cells reached a plateau phase ( 1.3-fold) after day 5 of the culture period (Fig.1d). Morphological observation at different time points revealed that IronQ increased the number of adherent cells by almost 90% con uence on Day 14 ( Fig. 1b) with an increasing number of the early outgrowth colonies (CFU-Hill) at day 7 post-IronQ treatment (Fig.1c). The CFU-Hill has been described by Hill et al., characterized by the colonies that displayed a central cluster of rounded and at cells with a radial arrangement of spindle-shaped cells (Fig.1c). These colonies were consistent with the endothelial progenitor cell (EPC) phenotype [30].

Cell population transition and characterization of PBMCs cultured under IronQ complex
To further characterize PBMCs expanded under the IronQ complex condition, surface expression of stem cell markers and markers related to angiogenesis was analyzed using ow cytometry. Based on the scatter diagram, PBMCs post-IronQ treatment (post-IronQ PBMCs) proportionally transition to a large cell population more common than in PBMC untreated control group (pre-IronQ PBMCs) (Fig. 2a). The red lines indicate the cellular-sized gates of lymphocyte and monocyte (R1) and the larger cell (R2). The proportion of each positive cell involved in the whole cells of (R1) and (R2) gates was estimated. The percentage of cells expressing endothelial lineage cells was signi cantly increased in CD105 and VEGF receptor 2 (VEGFR-2) in PBMCs post-IronQ treatment group whereas, there was no signi cant difference between the two groups in the number of cells expressing CD31.The percentage of monocytes/macrophages (CD14 and CD11b) was decreased in PBMCs post-IronQ treatment group versus the untreated control group. Besides, we observed a slight decrease in the stem cell marker CD34 in PBMCs post-IronQ treatment group (Fig.2b, c). Altogether, the augmented frequency for VEGFR-2 or CD105 was considerably higher in PBMC post-IronQ cells versus monocytes/macrophages (CD14 and CD11b). These ndings indicate that IronQ complex treatment promotes differentiation of circulating progenitor cells in peripheral blood into the pro-angiogenic cells. We also evaluate the dynamic changes of 7 different surface molecules during the culture of PBMCs treated IronQ complex. The result was showed in Fig. 2d. We found that the expression of angiogenic markers CD105 and VEGFR-2 was gradually increasing expressed, whereas expression of CD31 markers remained expressed at variable levels throughout the culture period. Not surprisingly, the pan leukocyte marker CD45 stabilized with culture time, but the stem cell marker CD34 also followed this pattern. The monocyte/macrophage markers were diminished during the culture. Interestingly, we observed that the marker expression was reached the peak on day 10 of the culture period.
PBMCs cultured with IronQ complex secrete vasculogenic, anti-in ammatory, and wound healing factors Conditioned medium (CM) by PBMCs post-IronQ treatment (post-IronQ PBMCs, at day 10) and untreated control PBMCs (control PBMC-CM) were evaluated for secreted angiogenic, anti-in ammatory, and wound healing factors. It appears that treatment with the IronQ complex stimulates the secreted paracrine factors from PBMCs. These molecules play a critical role in promoted angiogenesis and the wound healing process. The analysis of the amounts of secreted factors by enzyme-linked immunosorbent assay (ELISA). The results (Fig.3) revealed the presence of pro-angiogenic factors including, IL-8, matrix metalloproteinases 9 (MMP-9), and urokinase plasminogen activator (uPA), were signi cantly higher in post-IronQ PBMC-CM than in the control PBMC-CM group concurrently with the decrease of their inhibitor plasminogen activator inhibitor-1 (PAI-1). Inversely, the VEGF level was lower in post-IronQ PBMC-CM than in the control PBMC-CM. Additionally, the secretion of anti-in ammatory factor IL-10 was signi cantly CM still appear as a well-reorganized tube formation (Fig. 4a). We measured cumulative tube length at 24 h after seeding to estimate the stability of the tubular network. The quantitative result revealed a longer cumulative tube length in HUVECs cultured in post-IronQ PBMC-CM. Although, no difference in the total tube length of HUVECs cultured in EGM medium or control PBMCs-CM was observed (Fig. 4c). Moreover, HUVECs in post-IronQ PBMC-CM had signi cantly increased in the number of tubules per eld of view than EGM medium or control PBMC-CM (Fig. 4d). These ndings suggested that the treatment of PBMCs with IronQ secreted proangiogenic agents that support the tubular formation of endothelial cells.
To assess the angiogenic potential of the PBMCs treated with IronQ in vitro. These PBMCs were labeled with green uorescence (PKH-67) and then cocultured with HUVEC on Matrigel. IronQ-preconditioning PBMCs colocalized and rmly attached with endothelial tube network. They also participate at the junction point of the endothelial tubular network (Fig. 4b). These results revealed that when cocultured IronQ-preconditioning PBMCs that secreted angiogenic factors, preferential augment the tubular network, and these post-IronQ PBMCs were able to stabilize the networks. Although these expanded PBMCs did not form a network on their own.

Conditioned medium from PBMCs cultured under the IronQ complex (post-IronQ PBMC-CM) promotes migration of human umbilical cord vein endothelial cells
Since a tube formation involves the migration of HUVEC, we test the chemotactic response of HUVECs to post-IronQ PBMCs secreted factors using a Transwell migration assay. After 24 h, HUVECs migration in response to angiogenic factors secreted by IronQ-preconditioning PBMCs (post-IronQ PBMC-CM) was enhanced three to thirty-fold (P < 0.05) over migration in response to the condition medium from control PBMCs (Control PBMC-CM) or the negative control of 0.1% FBS medium alone. And the numbers of migrating HUVECs in the control PBMC-CM were signi cantly more than those in the negative control group. Not surprisingly, the highest number of migrating cells observed for the positive control group (10% FBS medium) had the greatest-promoting effect. (Fig. 5a, b).

Conditioned medium from PBMCs cultured under the IronQ complex (post-IronQ PBMC-CM) induces migratory ability of broblast cells
To investigate whether the PBMCs secreted factors affect the capacity of cell migration on broblast.
Wound closure migration assay was evaluated. L929 broblast cells were grown to con uency, then scratched and treated with ordinary medium or PBMCs secreted factors from condition medium qwwwwwwover the time course of 24 h. As shown in gure 6, the post-IronQ PBMC-CM induced a signi cantly shorter timeframe of L929 broblast migration, compared to control PBMC-CM or ordinary medium, at the time point of 1 and 3 h (Fig. 6b). After 9 h, scratched wound closure was almost completely ful lled in the L929 broblast cultures exposed to both of conditioned medium (post-IronQ PBMC-CM and Control PBMC-CM) compared with the ordinary medium, which showed about 75% closure then, and 100% closure after 24 h (Fig. 6a, b). This result indicated that post-IronQ PBMC-CM contained factors that promoted broblast cell migration.

Discussion
At present, cell-based therapy has become a great deal of interest in repairing ischemic damage. However, its clinical application has still limited due to the rarity number of regenerative cells. Another challenge facing cell-based therapy is identifying the outcome and effectiveness and real-time in vivo monitoring of the transplanted cells [31]. Fortunately, the development of molecular magnetic resonance imaging (MRI) technology provides new approaches for high sensitivity to studying transplanted cells' therapeutic effects by non-invasive dynamic monitoring [32]. Our previous studies investigated the novel MRI contrast agent synthesis and characterization from the Iron-Quercetin complex or IronQ. IronQ is a positive contrast for a T1-weighted MR image. The prominent characteristic of IronQ was the high e ciency of loading into the cells, and the magnetically labeled mononuclear cells were visualized by a clinical 1.5 T MR scanner when the cell quantity was more than 2,000 cells/µL. Interestingly, the effectiveness of visualizing the IronQ labeled cells were still detectable when the labeled cells were in the culture for 21 days. It has been shown that IronQ can act as a stimulating agent by favoring the proangiogenic cell differentiation of PBMCs. Moreover, IronQ is highly sensitive and has no toxicity but enhanced the therapeutic e ciency of labeled cells [29]. These results are suggesting that IronQ exerts the most outstanding excellent dual functions in applications of cell-based therapy.
In the present study, we have demonstrated that IronQ preconditioning enhanced in vitro function of human peripheral blood mononuclear cells (PBMCs). We found that culturing PBMCs under IronQ conditions were enriched with proangiogenic cells and blood-derived cells with regenerative capacity. Preconditioning PBMCs with IronQ enhanced the therapeutic potential of PBMCs, by secreting the greatest importance cytokines and growth factors supporting revascularization and tissue repair. The use of IronQ preconditioning produced an increase of a spindle-shaped cell type with a surface markers pro le characterized by proangiogenic cells, comprising, for example, CD31 + , CD105 + , and VEGFR-2 + cells. Freshly isolated PBMCs cultured in an endothelial growth factor condition for a short culture period of 4-7 days exhibited a spindle cell-like morphology with a mixed expression pro le of CD31, VEGFR-2, CD105, and von Willebrand factor, the so-called "early outgrowth endothelial progenitor cells" (early EPCs) or proangiogenic cells (PACs). Early EPCs contribute to angiogenesis mainly via paracrine signaling mechanisms. Early EPC secreted multiple proangiogenic cytokines and growth factors augment endogenous vessel growth, but they fail to differentiate into endothelial cells [33]. A recent study showed that transplantation of peripheral blood-derived early EPC positive for endothelial markers such as CD31, KDR (VEGFR-2), von Willebrand factor, and CD105 in patients with acute myocardial infarction. Clinical improvements have been observed in these therapeutic cells, including decreased infarct size and an increase in ejection fraction [34,35]. Culturing PBMCs under the IronQ condition also resulted in enhanced proliferation and PBMCs population transition. The results demonstrated that PBMCs cultured under the condition of IronQ doubled their population number in 7 days of culturing and still increased until Days 10, while the number of PBMCs in the control group progressively decreased with the increased time in culture ( Fig. 1, 2). The complex process of tissue repair involves a multistep, including angiogenesis and tissue regeneration, and is tightly controlled by the crosstalk between the cell populations in such a system [36]. Therefore, using a single cell type may be insu cient for successful treatment. There is growing evidence that angiogenesis is a critical process to tissue repair and regeneration. Dashtimoghadam et al., demonstrate that multifunctional cell therapy microcarriers contained mesenchymal stem cells (MSCs), endothelial cells, and vascular endothelial growth factor (VEGF) enhance the regeneration of bone tissue in vivo [37]. The new blood vessel formation is a prominent process during tissue repairing the damage. Recently, the study showed that combined use of stem cells and endothelial cells harness the therapeutic effect on cardiac damage regeneration than using stem cells or endothelial cells alone [38]. All together suggest that the pro-angiogenesis condition is a critical factor for promoting tissue regeneration.
Our study adopted a heterogeneous cell mixture model of mononuclear cells to allow for crosstalk between all components of PBMCs. We hypothesized that IronQ would stimulate the mononuclear cells to amplify cytokine signals and crosstalk between the signi cant cell population in such a system. The cytokine secreted from PBMCs treatment with IronQ are well known for their capacity to promote angiogenesis such as IL-8, MCP-1, uPA, MMP-9, and VEGF. These angiogenic factors are potent secreted from various monocyte-derived angiogenic cells, including EPCs, pericytes, monocytes, and macrophages cells [39][40][41]. Also, IronQ can stimulate mononuclear cells to produce the cytokine that contributes to regulating T cell differentiation, such as IL-10. This cytokine produced by M2 macrophages and induces Th2 and Treg lymphocyte functions [42]. M2 macrophages ameliorate anti-in ammatory and immunesuppressive phenotypes, and they promote angiogenesis and tissue repair. It is reported that intracellular iron status act as in modulation of macrophage plasticity and polarization. A recent study revealed the in uence of iron on innate immune and macrophage polarization in vivo and in vitro. The researchers found that an iron-rich status promotes M2 subtype macrophage and impaired M1 subtype activated response to LPS-induced pro-in ammatory [43]. Therefore, IronQ that contained iron might stimulate and load intracellular mononuclear cells resulting in PBMCs transform into alternative M2 macrophage cells. A result of cytokine secreted by M2 macrophage (IL-10) regulates T lymphocyte transform phenotypically polarized into their respective regenerative subsets (Th2 and Treg lymphocytes).
Interestingly, IronQ treatment can induce PBMCs to expand proangiogenic cells and change blood cells into the cells responsible for vascular and tissue regeneration. Through the IronQ treatment results of various combinations of therapeutic cells and secreted cytokines, we could conclude that cytokines play a synergistic role in the priming process. However, future experiments may involve the closer examination of responsible factors and cells to IronQ treatment for the generation of therapeutic cells.
Growing evidence from recent studies strongly suggests highlighting the paracrine effect of the transplanted cells in cell therapy [44]. In our research, IronQ preconditioning PBMCs could promote angiogenesis and tissue repair mainly via a paracrine manner. The conditioned medium analysis obtained from post-IronQ treated PBMCs contained a more incredible amount of several key proangiogenic cytokines, such as IL-8, uPA, MMP-9, MCP-1, TNF-alpha. Importantly, IL-8, which is a wellknown proangiogenic cytokine and plays a signi cant role in angiogenesis. Regarding their association with angiogenesis, the therapeutic target on IL-8 is currently under research for cancer therapy [45]. The angiogenic property of IL-8 is involved in endothelial cell proliferation and capillary tube organization [46]. Also, urokinase plasminogen activator (uPA), which increased more than 10-fold in post-IronQ PBMCs conditioned medium, plays a role in angiogenesis through proteolytic degradation of the extracellular matrix that facilities the subsequent proliferation and migration of endothelial cells [47]. This nding indicated that post-IronQ treated PBMCs were higher secreted proangiogenic factors. They may be re ected in our observation of enhanced tube formation and migration of HUVEC after treatment with condition medium from post-IronQ treated PBMCs. However, VEGF, one of the main proangiogenic growth factors, was not elevated but reduced in post-IronQ treated PBMCs, relative to control PBMCs. A negative feedback mechanism might explain the reduction of VEGF level through a high secreted TNF-alpha in post-IronQ treated PBMCs conditioned medium.
Tumor necrosis factor-alpha (TNF-α) is a secretory product of activated macrophages, a crucial proin ammatory mediator. In our nding, the secreted level of TNF-α slightly elevated in post-IronQ treated PBMCs, relative to control PBMCs. This nding indicates that post-IronQ treated PBMCs may bring proin ammatory cell populations more than control PBMCs. However, TNF-α has been implicated in angiogenesis during in ammation, wound repair, and tumor growth [48]. It has been reported that TNF-α mediates crosstalk between macrophages and ECs at sites of in ammation and enhanced and temporally regulated angiogenic sprouting begins [49]. These might be re ecting in our observed very early endothelial sprouting of HUVEC culture in Matrigel assay with the presence of culture medium from post-IronQ treated PBMCs, relative to conventional EGM medium. However, TNF-α may support angiogenesis via induced expression of proangiogenic genes, such as VEGFR-2, while blocking signaling through VEGFR-2. It results in delayed angiogenesis in the acute phase of in ammatory response. Therefore, TNF-α can exert pro-or anti-angiogenic effects depending on its expression during the angiogenic process [49]. Furthermore, TNF-α also is a potent broblast chemoattractant and stimulates broblasts proliferation and the local application of TNF-α in collagen-based biomaterials can improve promote healing of injured tissue [49][50][51]. Herein, IronQ-treated PBMCs secreted a higher level of TNF-α.
This phenomenon may be re ected in the ndings of enhanced migration of L929 broblast after cultured in post-IronQ treated PBMCs conditioned medium (Fig. 6). Moreover, in our study, the secreted of MMP-9, which playing a critical role in neovascularization and tissue remodeling for anti-brosis, were elevated in the post-IronQ treated PBMCs conditioned medium. Taken together, TNF-α, through its actions with MMPs, may play a role in improved wound healing. The recruitment and activation of monocytes/macrophages within the ischemic tissues are essential for the tissue repair process [52]. These secretory cytokines are known to be important in wound healing [53,54]. These results suggest that PBMCs treatment with IronQ complex promotes PBMCs to secrete high levels of growth factors and cytokines that are important for angiogenesis and the wound healing process. Following ischemic damage, the most frequently observed chemokines is monocyte chemotactic protein-1 (MCP-1). The MCP-1 is associated with monocyte recruitment and has been considered as one crucial proangiogenic factor [55]. A recent study in the hindlimb ischemic model has shown the excellent bene t of administering exogenous MCP-1 to increase blood ow to the ischemic tissue via the increase in monocyte/macrophage recruitment and augment the development of collateral neovascularization [56].
Furthermore, MCP-1 has been shown to mobilize and transdifferentiate the mononuclear monocyte lineage cells into endothelial-like cells [57].
The non-healing phenotype of chronic wounds is characterized by a lack of vascularization and wound re-epithelialization [58]. The wound healing process involves a multistep, including in ammation, angiogenesis, and tissue regeneration of the skin. These complex processes are tightly controlled by the interplay of different cell types in the wounded tissues, including in ammatory cells, broblasts, keratinocytes, and endothelial cells [59,60]. IronQ-treated PBMCs signi cantly enhance angiogenesis  obtained from PBMC to improve wound healing response in a murine model [65]. In pigs, the transplantation of endothelial progenitor cells (EPCs) was used to promote wound closure and angiogenesis. This work was published by Kado et al., who were able to enrich PBMC with EPCs and transplanted them into wounds performed on cyclosporine-immunosuppressed pigs [66]. Therefore, the combination of these data from the literature and our results contribute to consider IronQ-preconditioning PBMCs as a promising approach for treating chronic wounds.

Conclusions
The novel technique, IronQ-preconditioning PBMCs, investigated in this study, could be an alternative method for cell expansion and therapy for ischemic diseases and chronic wounds. It has the advantage of using paramagnetic agents to expand the limited number of progenitor cells from non-mobilized PBMCs without the addition of growth factors and serve as the magnetic label for MRI at the same time. However, further study is the warranty for a better understanding of the mechanism under the effect of IronQ on EPCs.